Method of assay

ABSTRACT

A kinetic assay method for quantifying an analyte in a sample.

The present invention relates to a method of assay for quantifying ananalyte in a sample, in particular to a kinetic assay method during thecourse of which a component of the assay system becomes at least partlybound, directly or indirectly, to the surface of a solid body.

The method of the present invention has particular applicability in thefield of immunoassays and will be described herein with particularreference to immunoassays; however the method can also be used in otherassays which rely on the affinity between the species to be assayed(hereinafter “ligand”) and a specific binding partner for the ligand(hereinafter “specific binding partner”).

The ligand concentration in such systems may be determined by monitoringthe extent of complex formation or rate at which complex formationoccurs. One preferred way of achieving this is by conjugating anadditional component having a measurable parameter to either the ligandor its specific binding partner. This additional component is known inthe art as a label and various chemical and biochemical labels are knowneg. radioisotopic or biochemiluminescent species, spin labels,fluorophores, chromophores, etc. In the course of such assays, the labelin effect becomes bound, indirectly at least, to a solid surface.

Conventionally, most immunoassay systems of the type described abovehave relied upon there being washing and/or separation step(s) in theassay protocol in order to separate bound label from label remaining insolution; the latter otherwise would be free to interfere with the boundlabel and lead to inaccurate results. Once separation has been effected,a variety of known techniques may be used to quantify the bound labeland thereby yield a measure of the concentration of ligand present inthe sample under investigation. In such systems, the separationprocedure must be repeated at each time (t) at which it is desired tomake a measurement, rendering the method as a whole somewhat labourintensive and slow. In addition to these problems, there is a degree ofarbitrariness in the estimation of the commencement of incubation of theassay which leads to errors in the overall timing.

In order to speed up reading the assay and/or to increase thesensitivity of conventional assay systems, it would be desirable to makekinetic measurements. The limitations referred to above mean thatconventional assay systems do not lend themselves to making reliablekinetic measurements and it has been done in only a very few cases wherethe characteristics of the assay system allow. For example, it is knownto make kinetic measurements in immunoassay systems in which enzymes arethe label of choice, the rate of evolution of the product of the enzymecatalysed reaction being the parameter which is measured. In this case,measurement of the enzyme label only shortens the duration of the signalgeneration step and has no impact on the time taken to measureantibody:antigen binding i.e. the kinetic measurement applies to thefinal step of the assay and not to the key immune reaction.

Kinetic measurements have also been used in certain immunosensors. Herethe rate of change of signal of the sample containing an unknownquantity of antigen is measured and compared with the same parameter forstandards containing known concentration of antigen. The most convenientway of achieving this is to construct a curve of rate of change ofsignal (dI/dt in measurands per unit time) versus known concentration ofantigen in the standard. In this way, dI/dt for the sample of interestmay simply be read off the standard curve to arrive at the unknownantigen concentration. Naturally, such a technique suffers the drawbackthat the assay must be allowed to attain an arbitrarily determinedequilibrium at which point a single end-point measurement of the signalis made. The speed with which equilibrium is reached may beprohibitively slow and this in itself can introduce errors in themeasured rate of change of signal which will be critically dependent onthe prevailing conditions (eg. temperature, viscosity). Clearly it isnot possible in such a system to obtain quick and accurate measurementsof the ligand concentration.

Kinetic measurements have also been used to determine the concentrationof an unknown in some immunoassays, as disclosed in EP-A-667528 (DaikinIndustries, Limited). However, such assays do not involve the continuousmonitoring of the concentration of the unknown.

In other assays systems, for example that disclosed in EP-A-184600(Battelle Memorial Institute) kinetic measurements may be made, but arenot used to determine the concentration of an unknown in a sample, withinstead a single final measurement being used in this regard.

The present invention is based on the finding that, in assay methods,during the course of which a component of the assay system becomesdirectly or indirectly bound to, or adsorbed on, the surface of a solidbody, a reliable measurement of said bound or adsorbed component (ie.without interference from the free component in solution) can beobtained by direct and continuous monitoring of said component.

It should be emphasised that the method of the invention relates toassay systems of both the direct and indirect variety, the onlyrequirement being that they involve the binding of a component of theassay system to the surface of a solid body. Direct assay methods maytypically involve monitoring the reflected and/or generated signalwithin an irradiated solid optical structure (eg. a waveguide) in orderto determine the extent to which (or the rate at which) the opticalcharacteristics of said optical structure and/or the generated signalare altered by the biochemical complexation of a ligand and specificbinding partner which is bound to said optical structure (eg.antigen/antibody complexation). Indirect assay methods may typicallyinvolve monitoring a label (eg. a fluorophore) bound to one or more ofthe components present in the assay and directly or indirectly to thesolid body. Such methods are described for example in inter aliaWO-A-88/07202 and WO-A-90/01166 (Ares Serono). The invention is equallyapplicable to displacement assays where the labelled component isremoved from the solid surface as a result of the antibody:antigeninteraction.

The novel assay method of the present invention has the advantage thatan indication of the unknown ligand concentration may be obtained at avery early stage of the incubation period without the need to wait forsome arbitrarily determined end-point such as equilibrium. Moreover, theoperator is able to observe the result continuously and judge whether itwould be worthwhile taking further readings in an attempt to improve theaccuracy of the result. Additionally, continuous monitoring allowsrandom errors caused by, for example, problems with instrumentation tobe readily identified. Any spurious result may simply be isolated andignored.

Thus in its broadest aspect the present invention provides the use ofkinetic measurements to determine quantitatively an unknown sample in anassay system in which a component thereof becomes at least partly bounddirectly or indirectly to the surface of a solid body, for example thesurface of a solid optical waveguide, electrode or piezoelectriccrystal.

By “kinetic measurements” are meant direct and continuous measurementsof a measurable property or effect associated with said bound component(hereinafter an “analyte dependent parameter”) at a time before theassay reaches a substantially steady state i.e. equilibrium.

Viewed from a further aspect, the present invention provides a method ofassay in which a component becomes at least partly bound, directly orindirectly, to a solid body, for example an optical waveguide, electrodeor piezoelectric crystal, characterised in that an analyte-dependentparameter associated with said component at said solid body is measuredin a direct and continuous manner and in that said measured analytedependent parameters are manipulated to quantitatively determine anunknown sample. In one embodiment the analyte dependent parameter is ananalyte dependent optical parameter (i.e. a measurable optical propertyor effect) but parameters relating to electrochemical or piezoelectricproperties/effects may be used.

In a further embodiment, the use or method according to the inventionmay be applied to an analyte of known concentration for the purposes ofcalibration.

In a particular preferred embodiment, said method comprises the stepsof:

(a) calibrating the assay system for x samples each of known analyteconcentration (C_(a)) by measuring continuously for each sampleindependently at a plurality of times (t_(y)) after the onset ofincubation the value of an analyte-dependent parameter (P_(z)),

(b) for an analyte of unknown concentration (C_(b)) measuringcontinuously n independent values of an analyte-dependent parameter(P_(d)) each at time t_(e) after the onset of incubation,

(c) combining the data (P_(d),t_(e)) from step (b) with the calibrationdata (P_(z), t_(y), C_(a)) from step (a) to calculate the unknown doseof analyte (C_(b)) at time t_(a).

The analyte-dependent parameter referred to above may conveniently beany parameter associated with the interaction between applied radiationand the relevant bound assay component and includes but is not limitedto light-absorbing, scattering, fluorescence emission, phosphorescenceemission, luminescence emission (including chemiluminescence,bioluminescence and electrochemiluminescence) or colour emissionproperties. The term is also intended to encompass the measurableeffects which the bound component may have for example on the refractiveindex or transmittability of the optical surface, on total internalreflection or surface plasmon resonance (SPR) within the solid opticalbody, or interactions with evanescent waves at the surface of the body.Devices and techniques for measuring such analyte dependent opticalparameters or manipulating the above-mentioned effects are known in theart.

The invention also extends to the use of nonoptical devices such aselectrochemical and other sensor devices (e.g. piezoelectric crystals).

As used herein, the term “solid body” is intended to refer appropriatelyto any of the known surfaces to which may usefully be bound ligandand/or specific binding partner components eg. in the form of anelectrochemical, optical, piezoelectric or fibre-optic biosensor asdescribed in U.S. Pat. No. 5,356,780 or an optical structure capable ofexhibiting an SPR effect (eg. a diffraction grating) or a transparentoptical body (eg. a prism, sheet or fibre acting as a waveguide such asis described in EP-A-170376 (Unilever)) of the type described inEP-A-171148 (Unilever) and WO-A-95/24632 (Applied Research Systems). Inelectrochemical assay devices where the solid body is an electrode, itis known to use components bound to magnetic beads or particles whichmay be attracted to a magnetic field created at the electrode. Thesedevices too are useful in the method of the invention and are describedin for example EP-A-170446 (Serono Diagnostics Limited).

In one embodiment of the invention, the solid body may be coated with aspecific binding partner to the analyte of interest. Specific bindingpartners may be coated onto the surface of the solid body by knowntechniques, for example, as described in EP-A-171148.

The invention is particularly suited to assay methods during the courseof which a component acting as a label and having optically measurableproperties such as light absorbing, light-transmitting, lightscattering, fluorescent, phosphorescent, luminescent or colourproperties becomes at least partly bound (directly or indirectly) to thesurface of a transparent solid body (eg. an optical waveguide),especially methods of the type described in, for example, EP-A170376 andEP-A-171148.

In embodiments of the invention which relate to indirect assaytechniques, the binding of labels directly or indirectly to one or otherof the ligand or its specific binding partner may be carried out bymethods well known to the skilled man. The identity of such labels issimilarly well-known to the skilled man and includes those mentionedhereinbefore.

The method according to the invention is, in certain embodiments,intended for use in specific binding assay procedures in chemical,biochemical or clinical test procedures, in particular to immunoassayprocedures. Examples of such procedures are described in inter aliaEP-A-0171148, WO-A-92/09892, WO-A-93/25892 and WO-A-93/25908.

The present method is also applicable to a wide variety of devicesprovided these are of a type which make use of a component bound to asolid body including, for example, dip-stick or test-strip sensors,devices using a “sample flow-through” configuration or devices employingsample containment. Sample containment devices are preferred forcarrying out the method of the invention, with a more preferred devicebeing a capillary fill device, especially a fluorescence capillarydevice, for example the type of device described in EP-A-171148,WO-A-90/14590 or in International patent application No. PCT/GB95/02236(Applied Research Systems ARS Holding NV Such capillary fill devices maybe used singly or in a suitable holder such as is described inWO-A-90/1830.

In carrying out the method according to the invention to determine anunknown sample, it is first necessary to calibrate the instrument usinga set of solutions containing known concentrations of analyte (ie. step(a) as defined hereinbefore). The protocol adopted for this step may beconveniently chosen by the operator and is in no way intended torestrict the scope of the invention. Such operator determined variablesinclude the number of standards (referred to as x above) which mighttypically be three or more, the number of devices, the number ofreadings, the time interval between readings and the total period overwhich calibration is carried out.

After filling a device with a particular standard, measurements of theanalyte dependent parameter (referred to more generally as P_(z) above)are taken at regular intervals, such as every five seconds, for as longas is appropriate. This procedure is optionally repeated for furtherdevices, yielding response data at each time point (referred to as t_(y)above) for all of the standard analytes. For each time point t_(y/A) itis, therefore, possible to produce a standard curve of P_(z) vs C_(a)(appropriate to time t_(y)). In one embodiment, the (P_(z), C_(a)) datamay be fitted to a standard equation such as an n parameter logisticequation, or appropriate algorithm, using any conventional fittingmethod such as a least squares method.

In step (b) of the method according to the invention, the unknownanalyte-dependent parameter (referred to as P_(d) above) may be measuredat any time-point (referred to as t_(e) above) and used to determine aconcentration by interpolation from the standard curve (P_(z), C_(a))for that time point. Appropriate smoothing software may be used toimprove the accuracy of the estimation of concentration of analyte inthe sample. Typically the (t_(e), C_(b)) data obtained in this step aremanipulated to give a dose versus time profile for the sample, anexample of which is given in FIG. 2.

As has previously been emphasised, the method according to the inventionis a kinetic method and in step (b) the interval between readings isoperator determined and is typically of the order of less than 60s,particularly less than 30s, especially less than 10s and more especially5s or less.

In practice, it is envisaged that the calibration data from step (a) ofthe method according to the invention may be prepared by a manufacturerfor each batch of reagents and presented as a series of standard kineticcurves. These curves would then be supplied to the customer viaconvenient means for storing machine readable encoded data such assoftware, bar codes or magnetic strips for each batch of reagents. Thus,for example, on running unknown samples, the appropriate instrumentwould carry the calibration curves in its software and use them as “lookup tables” in order to calculate the dose of analyte in the sample undertest.

Thus in a further aspect the present invention provides a method ofcalibrating an assay system comprising step (a) as hereinbefore definedand optionally thereafter fitting the (P_(z), C_(a)) data to a standardequation (appropriate to time t_(y)). A kit comprising an assay devicetogether with means for storing machine readable encoded data whichcontains calibration data P_(z), t_(y), C_(a) as hereinbefore definedand which is adapted to cooperate with reading means for the purpose ofquantitatively determining an unknown analyte forms a further aspect ofthe invention.

One technological area which has undergone significant advancement inrecent years is the so-called point-of-care assay systems. These rely onvery accurate, sensitive and rapid methods of assay to enable successfulnear patient testing to be performed. Clearly therefore the presentinvention, with the advantages referred to hereinbefore, lends itself tosuch technology.

The method of the invention is particularly applicable to assays ofantigens or antibodies, i.e. to immunoassays, and in one embodiment ofthe invention the ligand under assay is an antigen and the specificbinding partner comprises an antibody to the said antigen. However, asmentioned above, the invention is not to be taken as limited to assaysof antibodies or antigens. Examples of ligands which may be assayed bythe improved assay method of the invention are given in Table 1 below,together with an indication of a suitable specific binding partner ineach instance.

TABLE 1 Ligand Specific Binding Partner antigen specific antibodyantibody antigen hormone hormone receptor hormone receptor hormonepolynucleotide strand complementary polynucleotide strand avidin biotinbiotin avidin protein A immunoglobulin immunoglobulin protein A enzymeenzyme cofactor (substrate) or inhibitor enzyme cofactor enzyme(substrate) or inhibitor lectins specific carbohydrate specificcarbohydrate lectins of lectins

The method of the invention has very broad applicability but inparticular may be used in assays for: hormones, including peptidehormones (e.g. thyroid stimulating hormone (TSH), luteinizing hormone(LH), human chorionic gonadotrophin (hCG), follicle stimulating hormone(FSH), insulin and prolactin) or non-peptide hormones (e.g. steroidhormones such as cortisol, estradiol, progesterone and testosterone, orthyroid hormones such as thyroxine (T4) and triiodothyronine), proteins(e.g. carcinoembryonic antigen (CEA) and antibodies, alphafetoprotein(AFP) and prostate specific antigen (PSA)), drugs (e.g. digoxin, drugsof abuse), sugars, toxins, vitamins, viruses such as influenza,para-influenza, adeno-, hepatitis, respiratory and AIDS viruses,virus-like particles or microorganisms.

It will be understood that the term “antibody” used herein includeswithin its scope:

-   (a) any of the various classes or sub-classes of immunoglobulin,    e.g. IgG, IgA, IgM, or IgE derived from any of the animals    conventionally used, e.g. sheep, rabbits, goats or mice,-   (b) monoclonal antibodies,-   (c) intact molecules or “fragments” of antibodies, monoclonal or    polyclonal, the fragments being those which contain the binding    region of the antibody, i.e. fragments devoid of the Fc portion    (e.g. Fab, Fab′, F(ab′)₂), the so-called “half-molecule” fragments    obtained by reductive cleavage of the disulphide bonds connecting    the heavy chain components in the intact antibody or fragments    obtained by synthetic methods,-   (d) antibodies produced or modified by recombinant DNA techniques,    including “humanised antibodies”.

The method of preparation of fragments of antibodies is well known inthe art and will not be described herein.

The term “antigen” as used herein will be understood to include bothpermanently antigenic species (for example, proteins, peptides,bacteria, bacterial fragments, cells, cell fragments and viruses) andhaptens which may be rendered antigenic under suitable conditions.

The method of the present invention is applicable to the normal range ofsample types e.g. urine, serumbased and whole-blood samples, foodsamples such as water samples and milk samples and to the known range ofassay types, for example competition or sandwich assays including interalia direct antigen assays, competitive antigen assays, direct antibodyassays, sandwich antibody assays, linked antibody assays, competitiveantibody assays and the like.

The detailed preparation of the assay devices within the scope of themethod according to the invention and the assay procedures used tocollect the data are well known to the skilled man.

The invention will now be illustrated in a nonlimiting fashion by thefollowing Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the kinetic signal of a reaction standard.

FIG. 2 shows the results of the dependence of a measured dose on assaytime interpolated from a relevant standard.

EXAMPLES Example A

1. Preparation of starting materials:

1.1 Fabrication of antibody-coated optical waveguides:

Anti-PSA monoclonal antibodies were supplied by Serono Diagnostics S A,Coinsins, Switzerland. A sheet of Permabloc glass (Pilkington GlassLtd., St. Helens, UK) having a thickness of about 1 mm was cleaned withdetergent (eg. Tween 20) in ultra-pure water with ultrasonic agitation.The surface of the glass was activated by incubating it in a 2% solutionof aminopropyltrimethoxysilane in water (pH 3-4) for two hours at 75° C.After rinsing in water, the glass sheet was dried at 115° C. for atleast four hours. The glass was then incubated for 60 minutes in a 2.5%solution of glutaraldehyde in a 0.05M phosphate buffer (pH 7) and thenwashed thoroughly with distilled water. Anti-PSA antibody was patternedonto the glass by discretely dosing a 1% solution of the antibody inphosphate buffer (pH 7) onto the glass and incubating it for 2 to 4hours after which the glass sheet was washed with buffer solution.Unwanted adsorbed protein was removed by soaking with 6M urea solutionin a known manner. Finally a layer of sucrose/lactose was formed overthe surface of the glass sheet by spin coating. This formed plate 4 ofthe FCFD test device.

1.2 Preparation of PSA conjugated to allophycocyanin (APC):

A second anti-PSA monoclonal antibody, which recognises a differentepitope on the PSA molecule to the one used in 1.1 above, was conjugatedto allophycocyanin (λex=650 nm, λem=660 nm) by Molecular Probes Inc.,Eugene, Oregon, USA and was used as supplied.

1.3 Microdosing of the specific reagents over a discrete zone ofanti-PSA antibody:

An opaque coating was screen printed onto a clean sheet of Permablocglass as described in GB 8911462.3. The measurement zone of the devicewas fabricated by microdosing a layer of anti-PSA/allophycocyaninantibody conjugate in buffer containing polyvinyl alcohol in an area 3×7mm onto the glass over the zone. After the conjugate was air dried, alayer of polyvinyl alcohol (4% in buffer) was microdosed over theconjugate. Finally the whole sheet of glass was coated in a layer ofsucrose/lactose by spray coating. This formed plate 2 of the FCFD testdevice.

1.4 Fabrication of FCFD test devices:

FCFD test devices such as have been described in EP-A0171148 werefabricated by screen printing onto the waveguide resulting from 1.1above bonding tracks of an ultraviolet curing glue (UVS 91, NorlandInc., USA) containing glass microspheres of 100 μm diameter (JenconsLtd., UK) in a pattern defining the long edges of the capillary celldevices. A sheet of glass as defined in 1.3 above was then placed overthe waveguide and a vacuum applied to the laminate. As a result of thevacuum, the upper sheet of glass was caused to press down onto the glue,the glass microspheres defining a gap of 100 μm between the glasssheets. The laminate was then exposed to an ultraviolet light source tocure the glue. Finally, the laminate sheet was broken into individualtest devices as described in EP-A-0171148.

1.5 Apparatus used in the measurement of the PSA assay:

A simple fluorimetry apparatus comprising a continuous light source(provided by light emitting diodes which emit light at a suitablewavelength to excite the allophycocyanin fluorophore) and aphotomultiplier tube (PMT). Light emerging from the optical edge of theFCFD is filtered to remove stray pump light and the discrete angle rangerequired to read the bound fluorescence measured by focusing the lightonto the PMT through an aperture.

2. Assay Procedure for PSA:

Signals indicative of analyte concentration were obtained from the FCFDdevices by the following method. The device, containing the sample to beassayed, was flood illuminated with light appropriate to stimulate thefluorophore contained within the test reagentry. This input light iscontinuous and its intensity is repeatable at every required measurementtime point.

In estimating the concentration of an unknown sample, it is firstnecessary to calibrate the instrument using a set of solutionscontaining known concentrations of analyte. For the data presented here,seven standard concentrations were used, each concentration being run induplicate devices. After filling a device with a particular standardconcentration, measurements of the level of fluorescence were taken atregular intervals (every 5 seconds for the data presented). In this waythe kinetics of the reaction could be monitored, as demonstrated in FIG.1. Measurements were taken over a period of 20 minutes. After completingkinetic measurements on all devices, data was available (at all of thetime points) regarding the response to all the particular standardanalyte concentrations. It was therefore possible to produce a “standardcurve” corresponding to each of the time points. In the case of thepresent data the standard equation used was a four parameter logistic,having been fitted by a conventional least squares method.

Subsequent to this calibration procedure, samples of unknownconcentration were run in the same kinetics mode. The fluorescence levelat any time-point was interpolated off the associated standard curveenabling a concentration level to be ascertained. The results of thedependence of the measured dose on assay time interpolated from therelevant standard curve are shown in FIG. 2.

1. A method of assay in which a component becomes at least partly bound to a solid body characterised in that an analyte dependent parameter associated with said component is kinetically measured in a direct and continuous manner and the resulting measured analyte dependent kinetic data is continuously manipulated to continuously quantitatively determine an unknown sample for a period of time after the onset of incubation and before the assay reaches a substantially steady state.
 2. A method as claimed in claim 1 wherein said solid body is an optical waveguide.
 3. A method as claimed in claim 1 wherein said analyte dependent kinetic data is an optical parameter.
 4. A method as claimed in claim 3 wherein said optical parameter is fluorescence emission.
 5. A method as claimed in claim 4 wherein said solid body is in the form of a sample containment device.
 6. A method as claimed in claim 5 wherein said device is a capillary fill device.
 7. A method as claimed in claim 1 comprising the steps of (a) calibrating the assay system for a number x of samples, each of known analyte concentration (C_(a)), by measuring continuously for each sample independently at a plurality of times (t_(y)) after the onset of incubation the value of said analyte-dependent kinetic data (P_(z)), (b) for an analyte of unknown concentration (C_(b)) measuring continuously a number n of independent values of said analyte-dependent parameter (P_(d)) each at time t_(e) after the onset of incubation, (c) combining the data (P_(d), t_(e)) from step (b) with the calibration data (P_(z), t_(y), C_(a)) from step (a) to calculate the unknown dose of analyte (C_(b)) at time t_(e).
 8. A method as claimed in claim 2 wherein said kinetic data is fluorescence emission.
 9. A method as claimed in claim 1 wherein said solid body is in the form of a sample containment device.
 10. A method as claimed in claim 9 wherein said device is a capillary fill device.
 11. A method as claimed in claim 1 wherein said kinetic measurement, data manipulation and determination monitoring are continued until the assay is considered to have reached a substantial steady state.
 12. A method as claimed in claim 1 wherein said kinetic measurement, data manipulation and determination monitoring are discontinued before the assay reaches a substantial steady state.
 13. A method of assay in which a component becomes at least partly bound to a solid body, the assay having been calibrated for number x of samples, each of known analyte concentration (C_(a)), by measuring continuously for each sample independently at a plurality of times (ty) after the onset of incubation the value of an analyte-dependent parameter (P_(z)), characterized in that the method comprises the steps: for an analyte of unknown concentration (C_(b)) measuring in a direct and continuous manner a number n of independent values of an analyte-dependent parameter (P_(d)) which is associated with said component each at time t_(e) after the onset of incubation, and manipulating said measured analyte dependent parameter to continuously quantatively determine an unknown sample for a period of time after the onset of incubation and before the assay reaches a substantially steady state by combining the data (P_(d), t_(e)) with the calibration data (P_(z), t_(y), C_(a)) to calculate the unknown dose of analyte (C_(b)) at time t_(e). 